Method for modulating or controlling connexin 43(Cx43) level of a cell and reducing arrhythmic risk

ABSTRACT

A method of modulating or controlling connexin 43 (Cx43) level of a cell includes inducing mitochondrial reactive oxygen species (ROS) production in the cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part (CIP) application of U.S. application Ser. No. 12/929,786, Filed Feb. 16, 2011, which claims the priority benefit of U.S. Provisional Patent Application Ser. No. 61/305,668, filed Feb. 18, 2010, and is a continuation-in-part (CIP) application of U.S. application Ser. No. 12/289,005, filed Oct. 17, 2008, now U.S. Pat. No. 8,003,324B2, which claims the priority benefit of U.S. Provisional Patent Application Ser. No. 60/960,883, filed Oct. 18, 2007, all of the foregoing are hereby incorporated herein in their entirety by reference. This application further claims the priority benefit of U.S. Provisional Patent Application Ser. No. 61/503,096, filed Jun. 30, 2011, which is also hereby incorporated herein in its entirety by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The invention was made with government supported under grants RO1 HL1024025, T32 HL072742, P01 HL058000, RO1 HL106592, a VA MERIT grant, and an American Heart Association Midwest Affiliate Postdoctoral Fellowship # AHA10POST4450037. The government has certain rights in the invention.

FIELD AND BACKGROUND OF THE INVENTION

An increased level of angiotensin-II (AngII), as is found in heart failure, is associated with an increased risk of ventricular tachycardia (VT), and treatment with angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers reduces that risk (References 1 and 2). Investigating the mechanisms of AngII-induced arrhythmia may result in finding new antiarrhythmic targets. We created a mouse model of cardiac-restricted angiotensin converting enzyme (ACE) overexpression (Reference 3). We demonstrated that homozygous mice (ACES/8) have a high rate of sudden cardiac death (SCD), with telemetry monitoring showing that approximately 80% of the SCD resulted from VT and less commonly severe bradycardia (Reference 4). The VT and bradycardia were the result of c-Src tyrosine kinase activation, connexin43 (Cx43) reduction, and the impairment of gap junction conduction (References 4-6).

Excess amounts of reactive oxygen species (ROS) have been implicated in the genesis of arrhythmia (References 7-11). The level of ROS is elevated in ACE8/8 mice (Reference 5). ROS is known to activate c-Src (Reference 12). We sought to determine whether ROS mediated any of the Cx43 remodeling during renin-angiotensin system (RAS) activation. Despite considerable evidence that oxidative stress is arrhythmogenic, conventional antioxidants such as vitamin E have not produced impressive therapeutic results in clinical trials (Reference 13). The sources of ROS include the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activated by AngII and generally requiring the p67 subunit for activity, xanthine oxidase, uncoupled nitric oxide synthase (NOS) in part because of tetrahydrobiopterin depletion, and mitochondria (Reference 14).

In the present invention, the inhibitor therapies were tested to determine the major source of cardiac ROS contributing to arrhythmogenesis in ACE8/8 mice.

ASPECTS OF THE INVENTION

The present disclosure is directed to various aspects of the present invention.

One aspect of the present invention includes discovery and/or demonstration that mitochondrial ROS plays a role in arrhythmogenesis.

Another aspect of the present invention includes discovery and/or demonstration that mitochondrial ROS production is responsible for reduction in Cx43 level in cardiac cells.

Another aspect of the present invention includes discovery and/or demonstration that mitochondrial ROS production is responsible for adversely affecting or reducing ventricular gap function in cardiac cells.

Another aspect of the present invention includes discovery and/or demonstration that mitochondrial ROS production, through a signaling cascade, leads to a reduction in Cx43 level, thereby increasing arrhythmic risk.

Another aspect of the present invention includes discovery and/or demonstration that a mitochondria-targeted antioxidant, such as 2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO), restores the Cx43 to a normal level.

Another aspect of the present invention includes discovery and/or demonstration that a mitochondria-targeted antioxidant, such as 2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO), reduces arrhythmic risk associated with an altered Cx43 level.

Another aspect of the present invention includes discovery and/or demonstration that a mitochondria-targeted antioxidant, such as 2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO), reduces or prevents sudden cardiac death (SCD), or decreases or prevents ventricular tachycardia (VT) inducibility.

Another aspect of the present invention includes suggestions, discovery, demonstration, and/or development of possible therapeutic approaches, strategies, and/or treatment for arrhythmias associated with RAS activation states.

Another aspect of the present invention includes a method of modulating or controlling connexin 43 (Cx43) level of a cell, which includes inducing mitochondrial reactive oxygen species (ROS) production in the cell.

Another aspect of the present invention includes, in a cell having a reduced connexin 43 (Cx43) level, a method of raising the Cx43 level to a normal level, which comprises exposing the cell to an effective amount of a mitochondria-targeted antioxidant. The method can be carried out in vitro or in vivo.

Another aspect of the present invention includes, in a cell having an elevated mitochondrial reactive oxygen species (ROS) production, a method of reducing the ROS production, which comprises exposing the cell to an effective amount of a mitochondria-targeted antioxidant. The method can be carried out in vitro or in vivo.

Another aspect of the present invention includes a method of reducing arrhythmic risk associated with an altered connexin (Cx43) level of a cell, which includes administering a mitochondria-targeted antioxidant to a human or animal in need thereof. The method can be carried out in vitro or in vivo.

Another aspect of the present invention includes a method of preventing sudden cardiac death (SCD) associated with renin-angiotensin system (RAS) activation in a cell, which includes administering a mitochondria-targeted antioxidant to a human or animal in need thereof. The method can be carried out in vitro or in vivo.

Another aspect of the present invention includes a method of controlling or reducing inducibility of ventricular tachycardia (VT) associated with renin-angiotensin system (RAS) activation in a cell, which includes administering a mitochondria-targeted antioxidant to a human or animal in need thereof. The method can be carried out in vitro or in vivo.

Another aspect of the present invention includes a pharmaceutical composition, which includes 2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO) and a pharmaceutically acceptable carrier, diluent or excipient.

Another aspect of the present invention includes a therapeutic kit, which includes a pharmaceutical composition including 2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO) and a pharmaceutically acceptable carrier, diluent or excipient, and optionally instructions for the use thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

One of the above and other aspects, novel features and advantages of the present invention will become apparent from the following detailed description of the non-limiting preferred embodiment(s) of invention, illustrated in the accompanying drawings, wherein:

FIGS. 1 a-b illustrate that a mitochondrial antioxidant inhibits sudden cardiac death and ventricular arrhythmia inducibility. FIG. 1( a)—RAS-activation mice were treated with the following antioxidants: apocynin, L-NIO, sepiapterin, allopurinol, TEMPOL, and MitoTEMPOL. A group of ACE8/8 mice were also crossed with P67DN mice. Kaplan-Meier survival analysis and log-rank tests show significant improvement in the survival free from sudden arrhythmic death only in the ACE8/8 mice that were treated with MitoTEMPO. MitoTEMPO had no effect on wild-type mice (WT). FIG. 1( b)—Representative electrocardiograms (ECG lead II) and right ventricular electrograms (endocardial EGM) of WT, ACE8/8 and ACE8/8 mice treated with MitoTEMPO are shown. VT was induced in 90% of ACE8/8 mice (9 of 10) using a burst pacing protocol starting at 100 ms pacing cycle length (PCL) and decreasing to 30 ms PCL or 2:1 capture. Treatment with MitoTEMPO reduced VT inducibility in ACES/8 mice to 17% (one of six mice) using the same above pacing protocol (P<0.05);

FIG. 1 c illustrates that a general antioxidant did not improve survival. Kaplan-Meier survival analysis shows no improvement in the survival from sudden arrhythmic death in ACE8/8 mice treated with TEMPOL;

FIGS. 2 a-b illustrate that mitochondrial ROS is increased in RAS Activation. FIG. 2( a)—Mitochondrial ROS was measured using MitoSOX fluorescence. Representative confocal microscopy images show an increase in the mitochondrial superoxide level in ACE8/8 cardiomyocytes and suppression of that level with MitoTEMPO treatment. Flow cytometry analysis shows a 1.5 fold increase in the level of mitochondrial superoxide in ACE8/8 mice and MitoTEMPO decreased that level to normal. FIG. 2( b)—MitoTracker Green was used to quantify mitochondria. There is no significant difference among the control, ACE8/8 and ACE8/8 treated with MitoTEMPO groups (n=10 for each group, P=NS) in mitochondrial number;

FIG. 2 c illustrates that MitoTEMPO prevents activation of c-Src by RAS activation. Western blot analysis shows the level of phospho-(Tyr416) Src (active c-Src) is 32% higher in ACES/8 than in control mouse hearts (P<0.05), and reduces to the level of control after Mito-TEMPO treatment (n=5 for each group, P=NS);

FIG. 3 illustrates that RAS Activation was associated with mitochondrial injury. Electron microscopy shows damage to the inner membrane and cisterna of mitochondria and vacuous areas within mitochondria areas with RAS activation that are prevented by MitoTEMPO treatment. RAS activation did not significantly change the percent area occupied by mitochondria compared with the control (38±2%, 34±5%, 36±4% of cytoplasmic surface area, for control, ACE8/8, MitoTEMPO groups, respectively; P=NS), a finding consistent with the MitoTracker Green analysis;

FIGS. 4 a-b illustrate that a mitochondrial antioxidant recovers Cx43 in RAS-activation mice. FIG. 4( a) MitoTEMPO increases the total Cx43 level in ACE8/8 mice from 24% to 62% of the Cx43 level in the control mice (P<0.05). FIG. 4( b) Immunohistochemistry staining for Cx43 confirms the increase in Cx43 level in ACE8/8 mice by MitoTEMPO treatment;

FIG. 5 illustrates that Cx43 function is Improved with a Mitochondrial Anti-Oxidant. Cx43 functional assessment by the fluorescent dye diffusion technique reveals an increase in dye spread in ACE8/8 mouse hearts with MitoTEMPO treatment; and

FIG. 6 illustrates a proposed signaling cascade of RAS-induced arrhythmogenesis. Activation of AngII significantly increases mitochondrial ROS production which in turn activates c-Src and results in Cx43 reduction at the gap junctions. Impaired gap junction conduction provides substrate for ventricular arrhythmia and sudden arrhythmic death.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S) OF THE INVENTION

Previously, it was shown that a mouse model (ACE8/8) of cardiac renin-angiotensin system (RAS) activation has a high rate of spontaneous ventricular tachycardia (VT) and sudden cardiac death (SCD) secondary to a reduction in connexin43 (Cx43) level. Angiotensin-II activation increases reactive oxygen species (ROS) production, and these mice show cardiac oxidation. Here, it is determined that the source of ROS and that ROS played a role in the arrhythmogenesis.

In particular, the present invention is based, at least in part, on the discovery and/or demonstration that an ROS overproduction leads to a reduction in connexin 43 (Cx43) level in cardiac cells.

Wild-type and ACE8/8 mice with and without two weeks of treatment with L-NIO (nitric oxide synthase inhibitor), sepiapterin (precursor of tetrahydrobiopterin), MitoTEMPO (mitochondria-targeted antioxidant), TEMPOL (a general antioxidant), apocynin (NADPH oxidase inhibitor), allopurinol (xanthine oxidase inhibitor), and ACE8/8 crossed with P67 dominant negative mice to inhibit the NADPH oxidase were studied. Western blotting, detection of mitochondrial ROS by MitoSOX Red, electron microscopy, immunohistochemistry, fluorescent dye diffusion technique for functional assessment of Cx43, and in-vivo electrophysiology study were performed. Treatment with MitoTEMPO reduced SCD in ACE8/8 mice (from 74% to 18%, P=0.0005), decreased VT inducibility (from 90% to 17%, P<0.05), diminished elevated mitochondrial ROS to the control level, prevented structural damage to mitochondria, resulted in 2.6 fold increase in Cx43 level at the gap junctions, and corrected gap junction conduction. None of the other antioxidant therapies prevented VT and SCD in ACE8/8 mice.

Methods

The animal experiments were conducted according to the National Institutes of Health (NIH) Guide for the Care and Use of Experimental Animals and were approved by the University of Illinois Institutional Animal Care and Use Committee. A group of wild-type mice (n=10) with similar background to the ACE8/8 mice (C57BL), and the following groups of ACE8/8 mice were studied:

1. ACE8/8 mice untreated control (n=23).

2. ACE8/8 mice treated with 4′-hydroxy-3′ methoxyacetophenone (apocynin) (Sigma-Aldrich, St. Louis, Mo.) to inhibit the NADPH oxidase activity (Reference 15) (1.5 mmol/L in drinking water for two weeks, n=8).

3. ACE8/8 mice crossed with a P67 dominant negative (P67DN) mice to inhibit NADPH oxidase activity (n=10). P67 is an important subunit of NADPH oxidase (Reference 16).

4. ACE8/8 mice treated with N5-(1-iminoethyl)-L-ornithine, dihydrochloride (L-NIO) (Sigma-Aldrich) to inhibit nitric oxide synthase (NOS) (25 mg/Kg/d intraperitoneal injections for two weeks, n=10). L-NIO is an inhibitor of all NOS subtypes (Reference 17).

5. ACE8/8 mice treated with 2-amino-7,8-dihydro-6-(2S-hydroxy-1-oxopropyl)-4(1H)-pteridinone (sepiapterin) (Reference 18) (Sigma-Aldrich), a precursor of tetrahydrobiopterin, to prevent eNOS uncoupling without inhibition of NOS (5 mg/Kg/d intraperitoneal injections for two weeks, n=8).

6. ACE8/8 mice treated with 1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (allopurinol) (Reference 19) (Sigma-Aldrich) to inhibit xanthine oxidase (1 mmol/L in the drinking water for two weeks, n=10).

7. ACE8/8 mice treated with 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (TEMPOL) (Enzo Life Sciences), which is a general antioxidant and mimetic of superoxide dismutase (Reference 20) (2 mmol/L in drinking water for two weeks, n=8).

8. ACE8/8 mice treated with (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO) (Reference 21) (Enzo Life Sciences, Plymouth Meeting, Pa.) to target mitochondrial superoxide (0.7 mg/Kg/d intraperitoneal injections for two weeks, n=17).

In addition, a group of wild-type mice were treated with MitoTEMPO (0.7 mg/Kg/d intraperitoneal injections for two weeks, n=5) to evaluate for any possible harmful effects of treatment.

Survival Recording and Analysis

Survival of all treated and untreated groups were recorded every morning during the two weeks of treatment and/or observation. Survival was assessed by using Kaplan-Meier analysis and log rank tests.

Electrophysiology Study

For the electrophysiology studies, the control mice (n=5), ACE8/8 mice (n=10) and ACE8/8 mice treated with MitoTEMPO (n=6) were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (5 mg/kg). As it was previously described (Reference 4), after cutdown of the right internal jugular vein, a 1.1-F catheter with 0.5-mm inter-electrode spacing (EPR 800, Millar Instruments, Houston, Tex.) was placed into the vein and was advanced into the right ventricle. A constant current stimulator (A320, World Precision Instruments, Sarasota, Fla.) connected to a computer was used for cardiac stimulation. During the experiment, body temperature was maintained at 37° C. with a warming pad. Burst pacing at cycle lengths of 100 to 30 ms (or to the loss of 1:1 capture) was used to test for VT inducibility. A rhythm with more than three consecutive ventricular beats was considered to be VT.

Mitochondrial ROS Measurement by Confocal Microscopy

To measure mitochondrial ROS, the fluorescent probe MitoSOX Red was used as previously described (Reference 22). Briefly, cardiomyocytes were isolated from control, ACE8/8 or ACE8/8 mice treated with MitoTEMPO (n=3 for each group) as previously described (Reference 22). Cells were stained with 5 μM MitoSOX Red and 100 nM MitoTracker Green for 10 minutes at 37° C., followed by washing twice with warm MEM medium and were incubated for 10 minutes. Images were taken by a Zeiss LSM510 META confocal microscope (Carl Zeiss GmbH, Oberkochen, Germany) using an argon laser excitation (488 nm and 514 nm) with emission collection through a 560-nm long-pass filter. Images were captured using 63× water immersion objective lens at 1024×1024 pixels. Cell area was calculated, and the whole-cell fluorescence intensity of MitoSOX Red was measured with ImageJ software (NIH). The number of pixels of the cell fluorescence divided by the cell area was used to determine the mitochondrial ROS generation.

Mitochondrial ROS Measurement by Flow Cytometry

To quantify the mitochondrial ROS by flow cytometry, the measurements were carried out using Cyan ADP (Beckman Coulter, Brea, Calif.). Isolated cardiomyocytes from each group (n=3 animals for each group) were stained with 5 μM MitoSOX Red with a similar method as above. MitoSOX Red was excited by laser at 488 nm, a similar excitation (514 nm) used in confocal studies, and the data were collected for the FSC, SSC, pulse-width, and 585/42 nm (FL2) channels. Cell debris as represented by distinct, low forward and side scatter were gated out for analysis. The data are presented by histogram of mean intensity of MitoSOX fluorescence or fold change when compared with an unstained control with MitoSOX present.

Transmission Electron Microscopy

Control, ACE8/8 mice, and ACE8/8 mice treated with MitoTEMPO were studied (n=3 for each group). Tissues were washed with cold phosphate buffered saline (PBS), and fixed with EM Grade glutaraldehyde 4% in 0.1M cacodylate buffer (pH 7.4). Fixed tissues were incubated with osmium tetroxide 1% in cacodylate buffer for 2 h and processed for embedding. Ultra-thin sections were cut 83 nm, placed on 200 mesh copper grids, and stained with uranyl acetate and lead citrate. All materials were purchased from Electron Microscopy Sciences (Hatfield, Pa.). Samples were visualized using a JEM-1220 Jeol transmission electron microscopy (JEM, Peabody, Mass.), and micrographs were taken using a Gatan Digital Micrograph (Gatan Microscopy, Plesanton, Calif.). All microscopy measurements were performed using the University of Illinois Central Microscopy Research Core Facility. Random images (n=20) from each sample were taken for analysis. The area occupied by mitochondria and the ratio of the damaged area identified by white blank areas to the area of the whole mitochondrion were measured using a digital grid that was placed over each micrograph (15×15 grid with 225 points at cross sections). The points that fell within a mitochondrion were counted and were divided by the total number of points (i.e. 225 minus pseudospaces) to measure the area occupied by mitochondria. The total points of white areas within a mitochondrion were divided by the total points that fell within the mitochondrion to estimate the damage.

Western Blot Analysis

The control, ACE8/8, and ACE8/8 treated with MitoTEMPO mice (n=5 for each group) were sacrificed, and their hearts were excised. The ventricular tissue was homogenized in a buffer containing 20 mM of tris-(hydroxymethyl)-aminomethane (Tris-Cl) (pH, 7.4), 150 mM of sodium chloride (NaCl), 2.5 mM of ethylenediaminetetraacetic acid (EDTA), 1% Triton-100, 10 μL/mL of phenylmethylsulfonyl fluoride (PMSF), 10 μL/mL of protein inhibitor cocktail (Pierce, Rockford, Ill.), and 10 μL/mL of phosphatase inhibitor cocktail II (Sigma-Aldrich, St. Louis, Mo.). Protein samples (5 to 20 μg) were separated via 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to nitrocellulose membranes. The membranes were blotted with the primary antibodies against phosphorylated (Tyr 416) c-Src and Cx43 (Cell Signaling, Danvers, Mass.). For a loading control, the membranes were blotted with a primary antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotech, Santa Cruz, Calif.). After treatment with secondary anti-rabbit or anti-mouse antibodies, imaging was performed with enhanced chemiluminescence. The radiographic film images were scanned and analyzed with NIH ImageJ software. Cx43 and phospho-Src levels were corrected for the GAPDH level for each sample.

Immunohistochemistry

Control, ACES/8, and ACES/8 treated with MitoTEMPO mouse hearts (n=4 for each group) were fixed in 10% formalin. After which, 8-μm thick sections were blocked for 1 h at room temperature and then were incubated with anti-Cx43 antibodies (Cell Signaling) overnight at 4° C. at concentrations known to provide the best signal-to-noise ratio. The slides were reviewed with a Zeiss Axioskop microscope (Carl Zeiss, Inc, Thornwood, N.Y.), and photomicrographs with original magnification ×40 were taken from the apex, the mid-left ventricle (LV), and the LV base. From each of those sites, photomicrographs were taken from the endocardium and epicardium. The Cx43 content was quantified with the use of a grid that divided the field of view into 225 squares. At the intersection points aligning with the intercalated disks, Cx43 was scored as “1” (present) or “0” (absent). The results were expressed as the percentage occupied by Cx43 of the total area examined, excluding pseudospaces. This method has been used previously to quantify levels of collagen and Cx43 in cardiac tissue (References 4, 23 and 24).

Functional Assessment of Cx43

An established technique was used for measuring Cx43 function that involves fluorescent dye introduction and diffusion in intact heart muscle (References 4 and 25). Fresh hearts from control, ACES/8 and ACES/8 mice treated with MitoTEMPO (n=5 for each group) were obtained. A sample from each heart was placed in phosphate buffered saline at 37° C., the anterior surface of the left ventricle was punctured with a 27-gauge needle, and the sample was incubated with a droplet of 0.5% Lucifer yellow (LY) and a droplet of 0.5% Texas Red Dextran (TRD) in 150 mM of LiCl solution. After a 15-minute incubation, the samples were fixed in 4% formaldehyde for 30 min, washed in phosphate-buffered saline, frozen in liquid nitrogen, and sliced into 14-μm sections. The sections were mounted on microscope slides and examined on a Leica DM5000 B epifluorescence microscope (Leica Microsystems Inc., Bannockburn, Ill.). Digital images of the spread of LY and TRD were obtained. The measurement of the dye spread was performed with ImageJ software. Molecules of TRD are too large to traverse gap junctions and stain cells with disrupted sarcolemmal membranes. The TRD distribution was subtracted from the length of the LY spread at the same site to measure the true LY spread through gap junctions. Dye spread in longitudinal and transverse directions was assessed.

Statistical Analysis

The values are presented as the mean±the SEM. The t test, one-way analysis of variance with post hoc tests of significance, the Tukey honestly significant test, and the Fisher exact test for 2×2 tables were used where appropriate, and a P value of <0.05 was considered statistically significant. The survival data were analyzed with the Kaplan-Meier method, and the P value was calculated with the log-rank test. The correlation was assessed with the Pearson correlation coefficient method.

Results Mitochondria-Targeted Antioxidant Therapy Prevented Sudden Cardiac Death and Inducibility of Ventricular Tachycardia

The survival of all animals was recorded daily, and a survival analysis was performed with the Kaplan-Meier test. Only treatment with MitoTEMPO prevented SCD and improved survival in the ACE8/8 mice from 26% to 82% (95% Cl, 1.96 to 11.53; P<0.005). Although NADPH oxidase, xanthine oxidase, and uncoupled NOS are sources of increased cardiac ROS with RAS activation, none of the other antioxidant therapies was effective in prevention of SCD (FIG. 1 a). Treatment with TEMPOL, a general antioxidant, was not associated with improvement of survival free of sudden arrhythmic death (FIG. 1 c). Treatment of control mice with MitoTEMPO did not cause any death or gross abnormality in those mice.

In electrophysiology studies, VT was induced in 90% (nine of 10) of ACE8/8 mice with a mean pacing cycle length (PCL) of 44 ms. The induced VTs in the ACE8/8 mice were primarily monomorphic (88%); this suggests a predominant reentry mechanism for the VT episodes. VT inducibility was decreased from 90% to 17% (one of six) in ACE8/8 mice by MitoTEMPO treatment (P<0.05) (FIG. 1 by VT could not be induced in control mice.

RAS Activation Increased Mitochondrial Superoxide Levels

Quantification of mitochondrial ROS levels by the MitoSOX reduction and flow cytometry methods revealed a 1.5-fold increase in the mitochondrial superoxide level in the ACE8/8 mice compared to the control mice (p<0.05) (FIG. 2 a). MitoTEMPO treatment reduced mitochondrial ROS level to 1.1-fold of that in the control mice (P=NS) (FIG. 2 a). Quantification of mitochondria by MitoTracker Green did not show any significant change between those groups (FIG. 2 b).

MitoTEMPO Reversed Mitochondrial Damage in RAS Activation

The percent of the cytoplasmic area occupied by mitochondria was not statistically different among the groups studied, consistent with mitochondria quantification with MitoTracker Green (FIG. 3). Nevertheless, by electron microscopy, ACE8/8 mice showed significant damage to the mitochondria inner membrane and cisterna (FIG. 3). The damaged area identified by the ratio of vacuous area within a mitochondrion to the whole mitochondrion was significantly higher in the ACE8/8 than in the control mice. This ratio was ameliorated by MitoTEMPO treatment (6.5±3%, 15±4%, and 8.5±3% in the control, the ACE8/8, and the ACE8/8 mice treated with MitoTEMPO, respectively; P<0.05 for control compared to ACE8/8 mice). In electron microscopy study, rarely could gap junctions be identified in untreated ACE8/8 cardiomyocytes.

MitoTEMPO Increased Connexin43 Levels at the Gap Junctions

The total Cx43 level detected by Western blot was decreased in ACE8/8 mice to 24% of control (P<0.05), and MitoTEMPO treatment increased that to 62% of control (P<0.05) (FIG. 4 a).

Immunohistochemistry staining showed that the Cx43 level in ACE8/8 mice was decreased to a comparable level measured by Western blot, and most of the remaining Cx43 in the untreated ACE8/8 mice was no longer located at identifiable intercalated disks (FIG. 4 b). The level of active c-Src, phospho-(Tyr416), was 32% higher in ACE8/8 than in control mouse hearts, and it was reduced to that of control mice by MitoTEMPO treatment (P=NS compared to control) (FIG. 2 c).

MitoTEMPO Increased Gap Junction Conduction to the Control Level

Functional assessment of Cx43 was done by using a fluorescent dye diffusion technique (FIG. 5). The gap junction dye diffusion in longitudinal direction was reduced in the untreated ACE8/8 mice to 62% of that in the control mice (P<0.05). MitoTEMPO treatment returned the gap junction conduction to the normal range (P=NS compared to control).

Discussion

Cx43 is the major structural protein of ventricular gap junctions, and a significant decrease in Cx43 causes sudden death (Reference 26). Here, it is shown that cardiac RAS activation, as occurs in heart failure (References 1 and 27), was associated with a significant reduction in Cx43. This range of reduction in Cx43 is known to be arrhythmogenic (References 28 and 29). It was previously shown that c-Src mediates the reduction of Cx43 by AngII (Reference 4). The current experiments show that mitochondrial ROS plays a central role in the AngII-mediated Cx43 remodeling probably by ROS activation of c-Src (References 30 and 31).

Although ROS have been implicated in the genesis of arrhythmia, the best strategy to effectively reduce the level of ROS in order to prevent arrhythmia is not known. In this study, only a mitochondria-targeted antioxidant was able to prevent arrhythmia. Targeting other known ROS sources or using a general antioxidant was ineffective. This result, particularly the therapeutic difference between TEMPOL and MitoTEMPO treatments, suggests that AngII-mediated ROS production is highly compartmentalized within mitochondria in cardiomyocytes. It has been recently shown that AngII receptors exist on the mitochondrial inner membrane (Reference 32), and AngII may affect directly mitochondrial ROS production. In addition, an isoform of the NADPH oxidase (NOX4) exists in mitochondria (References 33 and 34), and AngII is known to activate NADPH oxidase (References 35 and 36). While our experiments do not suggest a role for the NADPH oxidase, NOX4 does not require P67 subunit for its activation (Reference 37), and apocynin may not effectively inhibit mitochondrial NOX4-dependent ROS production. Therefore, it is possible that this system could be involved in what appears to be AngII signaling directly to mitochondria without NADPH oxidase involvement, possibly through a ROS-induced-ROS mechanism (References 38 and 39).

This study does not preclude the possibility of other sources of ROS contributing to arrhythmogenesis in other cardiac pathological states. It has been shown that perfusion of the whole heart or isolated cardiomyocytes with H₂O₂ is arrhythmogenic, which highlights the importance of ROS production in arrhythmogenesis independent of the source of ROS (References 24 and 40). Our results may simply be a function of the relative amounts of the enzymatic ROS sources in a cardiomyocyte. Consistent with others (Reference 9), we found that more than 30% of the cardiomyocyte area was occupied by mitochondria, and these mitochondria were damaged by RAS activation. Similar findings of the importance of mitochondria as a source of ROS and accompanying mitochondrial damage were recently reported in other cardiac pathologies such as heart failure, a RAS activation state (References 9, 41 and 42).

Previously, it was shown that RAS activation reduced Cx43 and that gap junction impairment was the main substrate for slow conduction and ventricular arrhythmia (Reference 6). It was demonstrated that c-Src inhibition prevented Cx43 reduction and SCD (Reference 4). Here, it is shown that mitochondrial ROS inhibition reduced c-Src activation, suggesting a model of RAS induced activation, as shown in FIG. 6. The findings herein collectively can be explained by a signaling cascade where cardiac RAS activation increases mitochondrial ROS production and mitochondrial injury, activates c-Src, reduces Cx43 at intercalated disks through competition with activated c-Src, reduces gap junction function, and increases ventricular arrhythmias.

These results may have clinical implications in patients with heart failure because AngII and ROS are elevated (References 1, 27 and 43-46) and Cx43 is reduced in this condition, which is associated with increased sudden death (References 47 and 48). Moreover, the proposed signaling cascade could explain why angiotensin converting enzyme inhibitors and AngII receptor blockers decrease sudden death (References 1 and 44). In addition, these results may have clinical implications in pathological conditions with elevated levels of ROS and c-Src activation due to insults other than AngII, for example, in cardiac ischemia and in ischemia-reperfusion state (References 49 and 50). Nevertheless, a gene-targeted model of cardiac-restricted RAS activation was used, and the result may vary in systemic elevation of AngII. It is also possible that MitoTEMPO exerted part of its antiarrhythmic effects by mechanisms other than c-Src activation and Cx43 remodeling. On the other hand, the lack of ventricular fibrosis, a normal cardiac sodium current, and an unchanged ventricular effective refractory period in the ACES/8 mice at the age they were studied (References 3 and 5) support a major role for mitochondrial ROS in RAS-mediated Cx43 remodeling.

In summary, it was found that RAS activation resulted in mitochondrial injury, mitochondrial ROS production, a reduction in Cx43, and increased arrhythmic risk. These changes were ameliorated by a mitochondria-targeted antioxidant, but not agents targeted to other sources of cardiac oxidation or a general antioxidant. These results point out the importance of targeting specific ROS sources to increase therapeutic effectiveness and identify a possible new treatment for arrhythmias associated with RAS activation states.

From the above, it is concluded that mitochondrial oxidative stress plays a central role in angiotensin II-induced gap junction remodeling and arrhythmia, and that mitochondria-targeted antioxidants may be effective antiarrhythmic drugs in cases of RAS activation.

The invention also provides pharmaceutical or dietary supplemental compositions comprising 2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO). Accordingly, the compound (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO)), can be formulated for oral or parenteral administration for the therapeutic or prophylactic treatment of diseases or conditions associated with arrhythmogenesis, particularly associated with reduced connexin 43 level.

By way of illustration, the compound can be admixed with conventional pharmaceutical carriers and/or excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, wafers, and the like. Such pharmaceutical compositions contain from about 0.1 to about 90% by weight of the active compound (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO)), and more generally from about 10 to about 30%. The pharmaceutical compositions may contain common carriers and excipients, such as corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, and alginic acid. Disintegrators commonly used in the formulations of this invention include croscarmellose, microcrystalline cellulose, corn starch, sodium starch glycolate and alginic acid.

A liquid composition will generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in a suitable liquid carrier(s), for example ethanol, glycerine, sorbitol, non-aqueous solvent such as polyethylene glycol, oils or water, optionally with a suspending agent, a solubilizing agent (such as a cyclodextrin), preservative, surfactant, wetting agent, flavoring or coloring agent.

Alternatively, a liquid formulation can be prepared from a reconstitutable powder. For example a powder containing active compound, suspending agent, sucrose and a sweetener can be reconstituted with water to form a suspension; and a syrup can be prepared from a powder containing active ingredient, sucrose and a sweetener.

A composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid compositions. Examples of such carriers include magnesium stearate, starch, lactose, sucrose, microcrystalline cellulose and binders, for example polyvinylpyrrolidone. The tablet can also be provided with a color film coating, or color included as part of the carrier(s). In addition, active compound can be formulated in a controlled release dosage form as a tablet comprising a hydrophilic or hydrophobic matrix.

A composition in the form of a capsule can be prepared using routine encapsulation procedures, for example by incorporation of active compound and excipients into a hard gelatin capsule. Alternatively, a semi-solid matrix of active compound and high molecular weight polyethylene glycol can be prepared and filled into a hard gelatin capsule; or a solution of active compound in polyethylene glycol or a suspension in edible oil, for example liquid paraffin or fractionated coconut oil can be prepared and filled into a soft gelatin capsule.

Tablet binders that can be included are acacia, methylcellulose, sodium carboxymethylcellulose, poly-vinylpyrrolidone (Povidone), hydroxypropyl methylcellulose, sucrose, starch and ethylcellulose. Lubricants that can be used include magnesium stearate or other metallic stearates, stearic acid, silicone fluid, talc, waxes, oils and colloidal silica.

Flavoring agents such as peppermint, oil of wintergreen, cherry flavoring or the like can also be used. Additionally, it may be desirable to add a coloring agent to make the dosage form more attractive in appearance or to help identify the product.

The compounds of the invention and their pharmaceutically acceptable salts that are active when given parenterally can be formulated for intramuscular, intrathecal, or intravenous administration. A typical composition for intramuscular or intrathecal administration consists of a suspension or solution of active ingredient in an oil, for example arachis oil or sesame oil. A typical composition for intravenous or intrathecal administration consists of a sterile isotonic aqueous solution containing, for example active ingredient and dextrose or sodium chloride, or a mixture of dextrose and sodium chloride. Other examples of aqueous solution are lactated Ringers injection, lactated Ringer's plus dextrose injection, Normosol-M and dextrose, Isolyte E, acylated Ringer's injection, and the like. Optionally, a co-solvent, for example, polyethylene glycol; a chelating agent, for example, ethylenediamine tetracetic acid; a solubilizing agent, for example, a cyclodextrin; and an anti-oxidant, for example, sodium metabisulphite, may be included in the formulation. Alternatively, the solution can be freeze dried and then reconstituted with a suitable solvent just prior to administration.

The compounds of the invention which are active on rectal administration can be formulated as suppositories. A typical suppository formulation will generally consist of active ingredient with a binding and/or lubricating agent such as a gelatin or cocoa butter or other low melting vegetable or synthetic wax or fat.

The active compound is effective over a wide dosage range and is generally administered in a therapeutically effective amount. It, will be understood, however, that the amount of the compound actually administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered and its relative activity, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like. Suitable doses are selected to effect a blood concentration of about 100-300 μM, preferably 100 μM.

According to the invention, a compound can be administered in a single daily dose or in multiple doses per day. The treatment regimen may require administration over extended periods of time, for example, for several days, for from one to six weeks, or longer.

Suitable formulations for use in the present invention can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985).

The compositions of the present invention can be used to treat conditions associated with RAS activation, including all disease states and/or conditions that are acknowledged now, or that are found in the future, to be associated with the activity of connexin 43 levels. Such disease states include, but are not limited to, pathophysiological disorders, including hypertension, cardiac arrhythmogenesis, sudden cardiac death (SCD), ventricular tachycardia (VT), insulin-dependent diabetes, non-insulin dependent diabetes mellitus, diabetic neuropathy, seizures, tachycardia, ischemic heart disease, cardiac failure, angina, myocardial infarction, ventricular fibrillation, transplant rejection, autoimmune disease, sickle cell anemia, muscular dystrophy, gastrointestinal disease, mental disorder, sleep disorder, anxiety disorder, eating disorder, neurosis, alcoholism, inflammation, cerebrovascular ischemia, CNS diseases, epilepsy, Parkinson's disease, asthma, incontinence, urinary dysfunction, micturition disorder, irritable bowel syndrome, restenosis, subarachnoid hemorrhage, Alzheimer disease, drug dependence/addiction, schizophrenia, Huntington's chorea, tension-type headache, trigeminal neuralgia, cluster headache, migraine (acute and prophylaxis), inflammatory pain, neuropathic pain and depression.

In a preferred embodiment, the compound of the present invention is used to reduce arrhythmic risk, including heart failure, (SCD) and ventricular tachycardia (VT). As previously mentioned, arrhythmic risk is associated with a reduction in the connexin 43 level. As such, compositions comprising 2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO) can be administered to individuals in need of reduced arrhythmic risk to increase connexin 43 level, thereby reducing or preventing arrhythmic risk.

While this invention has been described as having preferred sequences, ranges, steps, materials, structures, components, features, and/or designs, it is understood that it is capable of further modifications, uses, and/or adaptations of the invention following in general the principle of the invention, and including such departures from the present disclosure as those come within the known or customary practice in the art to which the invention pertains, and as may be applied to the central features hereinbeforesetforth, and fall within the scope of the invention and of the limits of the appended claims.

REFERENCES

The following references, including those cited in the disclosure herein, are hereby incorporated herein in their entirety by reference.

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1. A method of modulating or controlling connexin 43 (Cx43) level of a cell, comprising the step of inducing mitochondrial reactive oxygen species (ROS) production in the cell.
 2. The method of claim 1, wherein the ROS production is induced by angiotensin.
 3. The method of claim 2, wherein an elevated angiotensin level causes overproduction of ROS.
 4. The method of claim 2, wherein angiotensin comprises angiotensin II.
 5. The method of claim 3, wherein an overproduction of ROS results in a reduction of Cx43 level.
 6. The method of claim 3, wherein an overproduction of ROS results in a reduction of Cx43 level at a ventricular gap junction.
 7. The method of claim 3, wherein, an overproduction of ROS results in a reduction of Cx43 level at intercalated disks.
 8. The method of claim 5, wherein a reduction in Cx43 level increases a risk for ventricular arrhythmia.
 9. The method of claim 6, wherein a reduction in Cx43 level reduces gap junction function.
 10. The method of claim 1, wherein ROS overproduction is reduced by a mitochondria-targeted antioxidant.
 11. The method of claim 10, wherein the antioxidant comprises 2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO).
 12. The method of claim 1, wherein the cell comprises a cardiac cell.
 13. In a cell having a reduced connexin 43 (Cx43) level, a method of raising the Cx43 level to a normal level, comprising the step of exposing the cell to an effective amount of a mitochondria-targeted antioxidant.
 14. The method of claim 13, wherein the antioxidant comprises 2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO).
 15. The method of claim 13, wherein the cell comprises a cardiac cell.
 16. In a cell having an elevated mitochondrial reactive oxygen species (ROS) production, a method of reducing the ROS production, comprising the step of exposing the cell to an effective amount of a mitochondria-targeted antioxidant.
 17. The method of claim 16, wherein the antioxidant comprises 2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO).
 18. The method of claim 16, wherein the cell comprises a cardiac cell.
 19. A method of reducing arrhythmic risk associated with an altered connexin (Cx43) level of a cell, comprising the step of administering a mitochondria-targeted antioxidant to a human or animal in need thereof.
 20. The method of claim 19, wherein the amount of the antioxidant is effective to reduce arrhythmic risk.
 21. The method of claim 19, wherein the amount of the antioxidant is effective to restore the Cx43 to a normal level.
 22. The method of claim 19, wherein the antioxidant restores the Cx43 level to a normal level by reducing or suppressing mitochondrial reactive oxidation species (ROS) production.
 23. The method of claim 19, wherein the antioxidant comprises 2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO).
 24. The method of claim 23, wherein the amount of antioxidant is effective to result in a more than two-fold increase in the Cx43 level.
 25. The method of claim 19, wherein the antioxidant is administered orally or intravenously.
 26. The method of claim 19, wherein the human or animal is suffering from arrhythmia.
 27. The method of claim 19, wherein the antioxidant comprises at least one member selected from the group consisting of a powder, a tablet, a capsule, a solution, a suspension, and an injectable formulation.
 28. The method of claim 19, wherein the cell comprises a cardiac cell.
 29. A method of preventing sudden cardiac death (SCD) associated with renin-angiotensin system (RAS) activation in a cell, comprising the step of administering a mitochondria-targeted antioxidant to a human or animal in need thereof.
 30. The method of claim 29, wherein the amount of the antioxidant is effective to reduce arrhythmic risk.
 31. The method of claim 29, wherein the amount of the antioxidant is effective to restore the Cx43 to a normal level.
 32. The method of claim 29, wherein the antioxidant restores the Cx43 level to a normal level by reducing or suppressing mitochondrial reactive oxidation species (ROS) production.
 33. The method of claim 29, wherein the antioxidant comprises 2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO).
 34. The method of claim 33, wherein the amount of antioxidant is effective to result in a more than two-fold increase in the Cx43 level.
 35. The method of claim 29, wherein the antioxidant is administered orally or intravenously.
 36. The method of claim 29, wherein the human or animal is suffering from arrhythmia.
 37. The method of claim 29, wherein the antioxidant comprises at least one member selected from the group consisting of a powder, a tablet, a capsule, a solution, a suspension, and an injectable formulation.
 38. The method of claim 29, wherein the cell comprises a cardiac cell.
 39. A method of controlling or reducing inducibility of ventricular tachycardia (VT) associated with renin-angiotensin system (RAS) activation in a cell, comprising the step of administering a mitochondria-targeted antioxidant to a human or animal in need thereof.
 40. The method of claim 39, wherein the amount of the antioxidant is effective to reduce arrhythmic risk.
 41. The method of claim 39, wherein the amount of the antioxidant is effective to restore the Cx43 to a normal level.
 42. The method of claim 39, wherein the antioxidant restores the Cx43 level to a normal level by reducing or suppressing mitochondrial reactive oxidation species (ROS) production.
 43. The method of claim 39, wherein the antioxidant comprises 2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO).
 44. The method of claim 43, wherein the amount of antioxidant is effective to result in a more than two-fold increase in the Cx43 level.
 45. The method of claim 39, wherein the antioxidant is administered orally or intravenously.
 46. The method of claim 39, wherein the human or animal is suffering from arrhythmia.
 47. The method of claim 39, wherein the antioxidant comprises at least one member selected from the group consisting of a powder, a tablet, a capsule, a solution, a suspension, and an injectable formulation.
 48. The method of claim 39, wherein the cell comprises a cardiac cell.
 49. A pharmaceutical composition, comprising 2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO) and a pharmaceutically acceptable carrier, diluent or excipient.
 50. A therapeutic kit, comprising a pharmaceutical composition comprising 2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO) and a pharmaceutically acceptable carrier, diluent or excipient, and optionally instructions for the use thereof. 